Severe sepsis, a clinical syndrome associated with infection, is a major complication that can develop after surgery or trauma, and kills approximately 225,000 persons annually. We have identified HMGB1, a protein known previously only as a transcription factor, as a late-acting pro-inflammatory cytokine mediator of sepsis, and have focused our efforts on studying its contribution to the panhogenesis of severe sepsis. The central objective of the NIH-supported studies during the previous funding period was to understand whether HMGB1 mediates beneficial or injurious responses in murine sepsis. We discovered that HMGB1 kills without causing shock, and that it is necessary for the lethal manifestations of sepsis in mice. High levels of HMGB1 are produced in humans and animals with sepsis, and administration of neutralizing anti-HMGB1 antibodies is significantly protective, even when antibody treatment begins twenty four hours after the surgical induction of sepsis. This clinically relevant delayed treatment approach has been shown effective with three independent strategies to inhibit HMGB1 release or to block its activity. These findings have raised critically important questions that will be explored in the proposed studies, to better delineate HMGB1 in the pathophysiology of sepsis. We now seek funding to continue this productive and important line of investigation of HMGB1 research. The Aims in the proposed studies will address the biological activity of HMGB1 in the serum of animals with sepsis, and study whether tissues of animals exposed to chronically elevated HMGB1 levels develop tolerance. After determining the biological activity of HMGB1 in four standardized bioassays (monocyte/ rnacrophages, whole blood, genetically engineered epithelial cells, and gastrointestinal epithelial cells) we will also determine the molecular and structural correlates of HMGB1 bioactivity. HMGB1 will be isolated from sepsis serum, and the amount of acetylation and other modifications characterized to reveal the biochemical requirements for biological activity.